Risk Factors of Hypovitaminosis D in HIV-Infected Patients on Suppressive Antiretroviral Therapy

Abstract

Low serum vitamin D levels are very common in human immunodeficiency virus (HIV)-infected patients. In our cross-sectional study, we investigated the association between 25-hydroxyvitamin D (25(OH)D) levels and serum inflammation markers [C-reactive protein (CRP), white blood cells (WBC), D-dimers, platelet count (PLT)] in 148 HIV-infected patients on combined antiretroviral therapy [28 on tenofovir alafenamide (TAF)] and 40 healthy controls. The controls were significantly older (56.6 ± 19.1 years for HIV(−) vs. 45.1 ± 11.8 years for HIV(+); p = .001) and more females were observed in this group (65% for HIV(−) vs. 16.7% for HIV(+); p = .001). The vitamin D serum level was comparable in the two studied groups (74.2 ± 35.9 nmol/L for HIV(+) vs. 78.0 ± 27.6 nnmol/L for HIV(−), p = .545). In HIV-infected group, a significant positive correlation between CD4+ cell percentage and vitamin D level was observed (r = 0.17; p = .036). Furthermore, the significant negative correlation between vitamin D level and CD8+ cell percentage, PLT, CRP, and D-dimers was seen. In univariate analysis, only TAF use and AIDS status was associated with vitamin D level deficiency. No other antiretroviral (ARV) drug nor gender or smoking had influence on vitamin D serum level. In multivariate analysis, only AIDS status and CRP level were correlated with vitamin D level (slope estimate = 11.6 and p = .032 and slope estimate = −0.83 and p = .002; respectively). In summary, we report that low vitamin D level may be associated with high CRP level in HIV-infected patients on suppressive antiretroviral therapy, especially in AIDS phase. More larger studies are required to assess our observation concerning TAF use and vitamin D level in HIV-positive patients.

Introduction

Human immunodeficiency virus (HIV) infection has become a chronic disease after introduction of combined antiretroviral therapy (cART). Nevertheless, even after complete viral suppression, HIV-infected patients have more comorbidities, such as cardiovascular and renal diseases, neurocognitive disorders, or osteoporosis in comparison to general population.¹ It has been shown that persistent systemic inflammation with hypercoagulation and monocyte activation as immune dysfunction may play a significant role in this multimorbidity.² Moreover, some inflammatory markers as high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), or soluble CD14, have been already linked with observed higher mortality in HIV-positive patients.³

Vitamin D is a prohormone mainly synthesized in the skin after exposure to sunlight. It can also be obtained from food or from special supplements. The 25-hydroxyvitamin D (25(OH)D) is the circulating form of vitamin D measured in patient serum. Its low level could be associated with osteoporosis, bone fractures, muscle weakness, diabetes, hypertension, cardiovascular diseases, and some cancers.^4

–7

Low serum vitamin D levels are very common in HIV-infected patients. It has been shown that risk of vitamin D deficiency is higher in HIV-positive black people, higher body mass index, older age, and being on cART, especially with efavirenz (EFV).^8
–10 Severe hypovitaminosis D may also correlate with current or past smoking and hepatitis C coinfection.¹¹ Negative interaction between vitamin D and hs CRP, IL-6, tumor necrosis factor (TNF)-alpha, and adiponectin serum level in HIV-infected patients has been already shown. Therefore, the potential association between vitamin D serum level and inflammation as metabolic dysregulation processes is underlined in this vulnerable population.^11
–13

In our cross-sectional study, we investigated the association between 25(OH)D levels and serum general inflammation markers (CRP, WBC, D-dimers, PLT) in well-controlled group of adult HIV-infected patients and healthy controls.

Materials and Methods

All HIV-infected patients who participated in this study were followed in the Department of Infectious and Tropical Diseases and Hepatology of Medical University of Warsaw, Poland. Inclusion criteria were as follows: Caucasian origin, age >18 years old, and undetectable serum HIV viral load from minimum 6 months of antiretroviral (ARV) treatment. HIV-negative controls were chosen from healthy volunteers from medical staff of the Department of Infectious and Tropical Diseases and Hepatology and from medical students of Medical University of Warsaw. All included participants provided written informed consent. No specific ethics approval was needed since blood sampling was performed as part of the usual care in a single center without additional visit or sampling. Fasting blood samples were taken from patients as standard biochemical, immunological, and virological evaluations. We measured plasma 25(OH)D, CRP, D-dimers, WBC, and PLT level among HIV-infected and HIV-negative individuals. Additionally, HIV infection duration, CD4 total count, CD4%, CD8 total count, CD8%, and CD4/CD8 ratio were assessed among HIV/AIDS patients. Moreover, data concerning smoking status were collected. Plasma 25(OH)D ranges were measured by luminescent method in VITROS 5600 Immunoanalyzer from Ortho-Clinical Diagnostics and defined as: 0–24.9 nmol/L—extremely low, 25.0–49.9 nmol/L—low, 50.0–74.9 nmol/L—moderate, 75.0–99.9 nmol/L—high moderate, 100.0–199.9 nmol/L—high, and >200.0 nmol/L—toxic level.

The R package was used for all statistical calculations. Appropriate statistical tests (Student's t-test, Welch's t-test, and Pearson's correlation) were used for each type of variable in the univariate analysis. In the multivariate analysis, a linear model with a stepwise elimination method guided by the p-value was used to determine which variables were independently associated with vitamin D levels. A p-value of <.05 was assumed to be statistically significant.

Results

One hundred and forty eight HIV-infected patients (78 with HIV infection, 70 with AIDS) and 40 HIV-negative controls were included in this cross-sectional study. Among HIV-infected patients and healthy volunteers males to females ratio were 125 versus 23 (84.5% vs. 16.7%) and 14 versus 26 (35.0% vs. 65.0%) with more females observed in volunteers group (65% for HIV(−) vs. 16.7% for HIV(+); p = .001), respectively. All HIV-infected patients were successfully treated with cART and had HIV serum viral load <50 copies/mL. The controls were significantly older (56.6 ± 19.1 years for HIV(−) vs. 45.1 ± 11.8 years for HIV(+); p = .001). The vitamin D serum level was comparable in the two studied groups (74.2 ± 35.9 nmol/L for HIV(+) vs. 78.0 ± 27.6 nnmol/L for HIV(−), p = .545). The baseline characteristics of all study participants are presented in Table 1.

Table 1.

The Baseline Characteristics of 148 HIV-Positive Subjects and 40 HIV-Negative Controls

	HIV-positive (mean ± SD) or % n = 148	HIV-negative (mean ± SD) or % n = 40	p
Age (years)	44.9 ± 11.9	56.6 ± 19.1	<.001
Percentage of males (%, n)	84.5%, 125	35.0%, 14	<.001
Percentage of smokers (%, n)	43.9%, 65	0%, 0	—
White blood cell count ( × 10⁹/L)	5.6 ± 2.1	6.2 ± 1.5	.163
Platelet count ( × 10⁹/L)	227 ± 76	208 ± 60	.139
D-dimers (ng/mL)	635 ± 958	296 ± 187	.027
CRP (mg/L)	16.1 ± 38.5	6.2 ± 3.8	.106
Length of HIV infection (years)	8.1 ± 5.8	—	—
Percentage of patients with AIDS (%, n)	47.3%, 70	—	—
CD4+ cell count (cell/μL)	465 ± 263	—	—
CD4+ cell percentage (%)	36.4 ± 16.5	—	—
CD8+ cell count (cell/μL)	760 ± 380	—	—
CD8+ cell percentage (%)	60.0 ± 16.2	—	—
CD4+/CD8+ ratio	0.76 ± 0.69	—	—
CD3+ cell count (cell/μL)	1262 ± 520	—	—
Percentage on NRTIs (%, n)	98.0%, 145	—	—
Percentage on TDF (%, n)	70.9%, 105	—	—
Percentage on TAF (%, n)	18.9%, 28	—	—
Percentage on rilpivirine (%, n)	19.6%, 29	—	—
Percentage on efavirenz (%, n)	16.2%, 24	—	—
Percentage on nevirapine (%, n)	4.7%, 7	—	—
Percentage on PIs (%, n)	37.1%, 55	—	—
Percentage on IIs (%, n)	25.0%, 37	—	—
Vitamin D serum level (nmol/L)	74.2 ± 35.9	78.0 ± 27.6	.545

CRP, C-reactive protein; HIV, human immunodeficiency virus; IIs, integrase inhibitors; NRTIs, nucleoside reverse transcriptase inhibitors; PIs, protease inhibitors; SD, standard deviation; TAF, tenofovir alafenamide; TDF, tenofovir disoproxil fumarate.

There was no difference in vitamin D level in male and female HIV-positive groups (mean 76.8 ± 32.7 nmol/L in males vs. 79.7 ± 32.2 nmol/L in females, p = .592). In HIV-infected group, a positive correlation between CD4+ cell percentage and vitamin D level was observed (r = 0.17; p = .036). Furthermore, a significant negative correlation between vitamin D level and CD8+ cell percentage, PLT, CRP, and D-dimers was seen (Table 2).

Table 2.

Association Between Vitamin D Levels and Quantitative Parameters (Univariate Analysis) in HIV-Positive Group

Quantitative parameter	r	R^2	p
Age (years)	−0.14	0.020	.086
Length of HIV infection (years)	−0.10	0.009	.250
CD8+ (cells/μL)	−0.07	0.004	.430
CD8+ (%)	−0.19	0.036	.020
CD4+ (cells/μL)	0.12	0.015	.141
CD4+ (%)	0.17	0.030	.036
CD3+ (cells/μL)	0.02	0.000	.814
CD4+/CD8+	0.13	0.018	.104
WBC ( × 10⁹/L)	−0.08	0.006	.36
PLT ( × 10⁹/L)	−0.2	0.039	.016
CRP (mg/L)	−0.28	0.081	.001
D-dimers (ng/mL)	−0.25	0.061	.002

PLT, platelet count; WBC, white blood cells.

In univariate analysis, only TAF use was associated with vitamin D level deficiency. TAF was used in 28 studied patients (18.9% of HIV-positive group) as a standard recommended part of ARV therapy, not because of renal impairment. Mean vitamin D level in tenofovir disoproxil fumarate (TDF)-treated group was 81.1 ± 34.2 nmol/L versus 65.3 ± 29.4 nmol/L in TAF-treated group, p = .026, respectively. No other ARV drugs nor gender or smoking had influence on vitamin D serum level (Table 3). However, in multivariate analysis, only AIDS status and CRP level were correlated with vitamin D level (Table 4).

Table 3.

Association Between Vitamin D Levels and Qualitative Parameters (Univariate Analysis) in HIV-Positive Group

Vitamin D level (nmol/L)	TDF USE	TAF USE	p
Vitamin D level (nmol/L)	mean (SD)	mean (SD)	p
Male gender	76.7 (32.7)	81.5 (40.0)	.537
AIDS	69.8 (31.6)	84.3 (34.5)	.009
NRTIs	77.4 (34.1)	78.3 (26.5)	.965
Rilpivirine	81.1 (33.4)	76.6 (34.1)	.520
Efavirenz	75.1 (28.7)	77.9 (34.8)	.714
Nevirapine	70.5 (22.2)	77.8 (34.3)	.582
PIs	78 (35.8)	77.1 (32.8)	.877
IIs	75.4 (36.5)	78.1 (33.1)	.666
Smoking	74.8 (36.6)	79.5 (31.6)	.415

Table 4.

Variables That Had a Statistically Significant Association with Vitamin D Levels in the Multivariate Model

Parameter	Parameter level	Estimated change in Vitamin D level	p
AIDS status	No AIDS	Reference level	.032
AIDS status	AIDS	−11.6 nmol/L	.032
CRP	1 mg/L increase	−0.83 nmol/L	.002
Others	Not statistically significant		>.05

The model included every parameter from Tables 2 and 3.

Discussion

In our study, vitamin D serum levels did not differ between HIV-positive patients and the control group. The vitamin D deficiency occurrence in HIV-positive and HIV-negative group was similar and nonstatistically significant (24.3%, n = 36 vs. 17.5%, n = 7; p = .586).

By contrast with the results of the previous studies we observed no correlation between vitamin D level and age, gender, length of HIV infection, or smoking status in our HIV-positive group.^8,11,14 It has been shown that strong risk factor for vitamin D deficiency in the HIV population is ARV regimen, EFV, and protease inhibitor (PI) use.^15,16 However, recently Benguella et al. showed negative correlation between hypovitaminosis D and raltegravir-based cART. Moreover, they also observed positive correlation between EFV and boosted PI withdrawal and normalization of 25(OH)D vitamin level.¹⁴ In our study, we did not show the influence of EFV, PIs, integrase inhibitors, or non-nucleoside reverse transcriptase inhibitors (NNRTIs) use on vitamin D levels. However, for the first time, we observed the significantly lower vitamin D levels in patients treated with tenofovir alafenamide (TAF) in comparison to the TDF group (p = .026). Several studies showed that TDF is associated with reduction in bone mineral density (BMD) and switching from TDF to TAF led to improved BMD.¹⁷ TDF may have an influence on the relationship between Vitamin D and parathyroid hormone (PTH).¹⁸ Vitamin D deficiency in persons treated with TDF is associated with hyperparathyroidism.¹⁹ Havens et al. showed that this high PTH serum concentration was caused by TDF influence on higher vitamin D binding protein and lower free 1,25-OH(2)D, leading to a functional vitamin D deficiency.²⁰ To our knowledge, there is no information concerning TAF use and hypovitaminosis D risk in HIV-infected population. In our study, TAF was associated with vitamin D level deficiency only in univariate analysis what may suggest that there are other factors correlated with TAF-vitamin D level axis or our studied group was too small. As a result, larger longitudinal studies should be done to confirm our findings.

It has been shown that vitamin D plays a role in the innate and adaptive immune systems as influences on immune restoration after cART initiation.^21
–23 In our study, we showed significant positive correlation between vitamin D level and CD4 percentage and negative correlation between CD8 percentage. Moreover, AIDS status was independent of hypovitaminosis D risk factor in univariate as in multivariate analysis. Our results may be vicariously linked with Shepherd's study where the association between CD4 cell count and vitamin D level had been found, suggesting that vitamin D could be a cofactor of CD4 restoration and proliferation.²⁴

Finally, we showed that vitamin D level is negatively correlated with several markers of inflammation, including CRP, D-dimers, and PLT in HIV-positive individuals. However, in our group of patients, only high CRP level and AIDS status were independent risk factors of low vitamin D level in multivariate analysis. In recent years, several studies presented contradictory results concerning inflammation markers and hypovitaminosis D in HIV-infected individuals. Some of them showed correlation between hsCRP, TNF alpha, or IL-6 and vitamin D level in HIV-infected subjects, and some did not.^11,13,25,26 It has been shown that the immune activation is lower in HIV-infected patients with cART-induced viral suppression.²⁷ All our studied subjects had undetectable HIV viral load. However, they had significantly higher D-dimer level in comparison to controls. Since all patients were successfully treated, we suggested that these inflammatory parameters might be increased due to other comorbidities (AIDS-defining or non-AIDS-defining) presented during the sample collection for the study. However, we recommend vitamin D serum level measurement in all HIV-infected patients, especially with elevated CRP level because of high risk of vitamin D deficiency.

The limitations of our study should be noted. These include relatively small size of studied groups. Moreover, HIV-infected group and controls were not compatible with age and gender. The other important limitation is its retrospective nature and lack of data concerning vitamin D supplementation and coinfection with HBV or HCV status. Finally, all studied subjects were Caucasians.

In summary, we report that low vitamin D level may be associated with high CRP level in HIV-infected patients on suppressive antiretroviral therapy, especially in AIDS phase. More larger studies are required to assess our observation concerning TAF use and vitamin D level.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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